Remove the supernatent and resuspend cell pellet in 0.3 mL (0.6 mL) Solution I. Gently vortex or pipet up and down to resuspend.

Add 0.3 mL of Solution II and gently mix. Incubate at room temperature for 5 minutes. Note: The appearance of the suspension should change from turbid to almost translucent.

Slowly add 0.3 mL of solution 3, mixing gently during addition. A thick white percipitate of protein and E. coli genomic DNA will form. Place the sample on ice for 5-10 minutes.

Centrifuge for 10 minutes at 14,000 x g.

Gently transfer the supernatent to a microcentrifuge tube containing 0.8 mL of isopropanol. Do not transfer any white percipitate. Invert the tube a few times to mix and place on ice for 5-10 minutes. Proceed directly to Step 9 or store sample at -20 C overnight.

Centrifuge the sample for 15 minutes at 14,000 x g at room temperature.

Carefully remove the supernatent, taking care not to disturb the pellet. Add 0.5mL of cold 70% ethanol. Invert the tube several times to wash the pellet.